Abstract

The APC/Cdh1 E3 ubiquitin ligase plays an essential role in both mitotic exit and G1/S transition by targeting key cell-cycle regulators for destruction. There is mounting evidence indicating that Cdh1 has other functions in addition to cell-cycle regulation. However, it remains unclear whether these additional functions depend on its E3 ligase activity. Here, we report that Cdh1, but not Cdc20, promotes the E3 ligase activity of Smurf1. This is mediated by disruption of an autoinhibitory Smurf1 homodimer and is independent of APC/Cdh1 E3 ligase activity. As a result, depletion of Cdh1 leads to reduced Smurf1 activity and subsequent activation of multiple downstream targets, including the MEKK2 signaling pathway, inducing osteoblast differentiation. Our studies uncover a cell-cycle-independent function of Cdh1, establishing Cdh1 as an upstream component that governs Smurf1 activity. They further suggest that modulation of Cdh1 is a potential therapeutic option for treatment of osteoporosis.

A. Immunoblot (IB) analysis of whole cell lysates (WCL) derived from 293T cells transfected with HA-Smad1 together with Myc-tagged Smurf1 in the presence of increasing amounts of Myc-Cdh1 or Flag-CKIP1. Myc-Cdc20 was included as a negative control.B. Immunoblot (IB) analysis of whole cell lysates (WCL) derived from 293T cells transfected with HA-MEKK2 together with Flag-tagged Smurf1 in the presence of increasing amounts of Myc-Cdh1 or Flag-CKIP1.C. HeLa cells were infected with a shSmurf1 lentiviral vector (with shGFP vector as a negative control) for 24 hours and then selected with 1μg/ml puromycin for 72 hours to eliminate the non-infected cells. The resulting cell lines were transfected with HA-MEKK2 in the presence of increasing amounts of Myc-Cdh1. 40 hours post-transfection, whole cell lysates (WCL) were collected to probe with the indicated antibodies.D. Purified recombinant GST-Cdh1 augments the E3 ligase activity of Smurf1 to ubiquitinate Smad1 in vitro. Bacterially expressed and purified His-Smad1 and GST-Smurf1 were incubated with the indicated, purified recombinant GST-Cdh1 or GST-CKIP1 proteins, purified E1, E2 and ubiquitin as indicated at 30° C for 60 minutes. The ubiquitination reaction products were stopped by the addition of 8 M urea followed by His-tag pull-down in the presence of 8 M urea to eliminate any possible contamination from Smad1-associated proteins. The reactions were then resolved by SDS-PAGE and probed with the indicated antibodies.(see also )

A. Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with Flag-Smurf1, Myc-Smurf1 and the indicated HA-tagged Cdh1, CKIP1, Smad1 or Smad7 constructs. Thirty hours post-transfection, cells were pre-treated with 10 μM MG132 for 10 hours to block the proteasome pathway before harvesting.B. Autoradiography of 35S-labelled Smurf1 bound to the indicated GST fusion proteins in the presence of increasing amounts of 35S-labelled Cdh1.C. Autoradiography of 35S-labelled Cdh1 bound to the indicated GST fusion proteins.D. Deletion of the C2 domain augments Smurf1 auto-ubiquitination in vitro. Various bacterially expressed and purified GST-Smurf1 proteins were incubated with purified E1, E2 and ubiquitin as indicated at 30° C for 15 minutes. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the indicated antibodies.E. Deletion of the C2 domain augments the E3 ligase activity of Smurf1 towards ubiquitinating RhoA in vitro. Bacterially expressed and purified His-RhoA and the indicated GST-Smurf1 proteins were incubated with purified E1, E2 and ubiquitin as indicated at 30° C for 60 minutes. The ubiquitination reaction products were stopped by the addition of 8 M urea and subsequent His-tag pull-down was performed in the presence of 8 M urea to eliminate any possible contamination from RhoA-associated proteins before being resolved by SDS-PAGE and probed with the indicated antibodies.(see also )

A. Immunoblot analysis of HCT116-WT or HCT116-p53-/- cells infected with the indicated lentiviral shRNA constructs. The infected cells were selected with 1 μg/ml puromycin for 72 hours to eliminate the non-infected cells before harvesting for immunoblot analysis.B. Immunoblot analysis of primary mouse calvarial osteoblast cells infected with the indicated lentiviral shRNA constructs. The infected cells were selected with 1 μg/ml puromycin for 72 hours to eliminate the non-infected cells and the resulting cells were cultured for 6 days under osteoblast differentiation media before harvesting for immunoblot analysis.C. Immunoblot analysis of primary murine Cdh1F/F calvarial osteoblast cells, which were derived from neonatal Cdh1F/F mice, infected with a lentivirus encoding Cre to deplete endogenous Cdh1 (empty vector infected cells were used as negative controls).D. Immunoblot (IB) analysis of T98G cells infected with the indicated lentiviral shRNA constructs together with the indicated pBabe-Hygro-Cdh1-expression vector. The infected cells were selected with 1μg/ml puromycin and 200 μg/ml hygromycin for 72 hours to eliminate the non-infected cells before harvesting for immunoblot analysis.E. Immunoblot analysis of immortalized WT or Smurf1-/- mouse calvarial osteoblast cells infected with the indicated lentiviral shRNA constructs. The infected cells were selected with 1 μg/ml puromycin for 72 hours to eliminate the non-infected cells before harvesting for immunoblot analysis.(see also )

Depletion of Cdh1 leads to inactivation of Smurf1 and augmentation of the MEKK2 signaling pathway to induce osteoblast differentiation

A-C. Depletion of Cdh1 in primary mouse calvarial osteoblasts promotes osteoblast differentiation. Primary mouse calvarial osteoblast cells were derived from neonatal mice and infected with the indicated lentiviral shRNA constructs. The infected cells were selected with 1 μg/ml puromycin for 72 hours to eliminate non-infected cells and the efficiency of Cdh1 depletion was examined by anti-Cdh1 immunoblot analysis in (A). Afterwards, the generated cells were incubated in osteoblast differentiation medium for 21 days before the Fast Blue staining for ALP activity (shown as blue staining) and von Kossa staining for minerilization (shown as black staining) in (B). Additionally, the Cdh1 knockdown efficiency and the expression of various characteristic osteoblast markers were determined by real-time PCR analysis in (C). Three sets of independent experiments were performed to generate each data point in (C) and the error bars represent means ± SD.D-E. The osteoblast differentiation is also increased in Cre-encoding lentivirus-induced Cdh1 depletion in primary Cdh1F/F calvarial osteoblast cells (empty vector (EV) lentivirus was used as a negative control). The infected cells were selected with 1 μg/ml puromycin for 72 hours to eliminate non-infected cells, the resulting cells were then incubated in osteoblast differentiation medium for 21 days before Fast Blue staining and von Kossa staining in (D). Cre-encoding lentivirus-induced Cdh1 depletion and the expression of various characteristic osteoblast markers were monitored by real-time PCR analysis in (E). Three sets of independent experiments were performed to generate each data point in (E) and the error bars represent means ± SD.F. Ectopic expression of WT-Cdh1 and ΔC-box-Cdh1, but not N174-Cdh1 could suppress the increased osteoblast differentiation phenotype in Cdh1-depleted primary calvarial osteoblast cells. Primary calvarial osteoblast cells derived from Cdh1F/F mice were co-infected with Purolentivirus encoding Cre (or a lentiviral vector encoding EGFP as a negative control) and the indicated Hygro-lentiviral vector encoding WT-Cdh1 and various Cdh1 mutants. The infected cells were selected with 1 μg/ml puromycin and 200 μg/ml hygromycin for 96 hours to eliminate non-infected cells. Afterwards, the generated cells were incubated in osteoblast differentiation medium for 21 days before von Kossa staining.(see also )